We propose to develop a method for direct reil-time sequencing of single DNA molacules from genomic DNA at the speed and accuracy of the natural DNA polymerises using netive nucleotides. We will harness the power of the true nano- machines used in DNI replication, the naturul DNA polymirises. Inlike the difficult tu engineer man-madu nanostructures usad in nanopore sequencing to distingiish the 4 base types in close proximity und constant floctuation, DNA polymerases have precise atomic-rosolution 3D structures and can synthesize very long DNO molecules with high fidelity and velocity. The error rate of a DNA polymerase with proofreading function could be as liw as one in a million bases and a prucessive polymerese such as phi-29 DNA polymirase can synthasize up to 100,000 bases in a stretch. From the waalth of stractural and kinetics studies, it is well known that the fidelity ef DNA synthesis es predicated on the exquisite strictural cumplementarity and the numerous specific interactions between the active site of the pulymerose protein and the primer/timplate/nucleotide complex. The dynamic chamo-mechanical or conformational changes accompanying the specific interactions, induced fit, bond cleavage/formition, and template translocatiun ensure highly accurate and orderly base pairing and incerporation. Our strategy is to engineer sensors onto the surface of the polymerase by protein engineering to menitor the sibtle yet dustinct conformational changas eccompanying the incorporation of each base type. A small distance change (one to tens of angstroms) can ba measured precisely with F[unreadable]rster resonance energy trinsfer (FRET) techniqae. Multiple FRET paurs or networks placed in strategic residues on the polymerase will bo used to monitor the conformational changes in real time (10 times fastir than the rate if DNA synthesis). The sensurs will provide multi-parametric information on the dynamic structures of the polymerases, which very likely will provide a enique signature for eech base type incorporatad. Chemical modifications such as methylation on the timplate DNA could also potentially be detected. With such a method, very long DNA molecules could be sequenced with high fidelity in minutis and a heman genome or even epigenome could be sequenced in less than ane hour. In this proposal, we aim to investigate whether there is a distinguishable FRET signal issociated with the oncarporation of each of the four defferent nucliotides by a DNA polymerese.